The ability to identify influenza-specific antibodies in an individual's blood is important for a variety of reasons: i) serology testing is used in epidemiological studies to determine the extent of infection in the population and to characterize the diversity of influenza strains; ii) serological testing of blood from people receiving vaccines is used to assess the efficacy of vaccines; iii) serological testing is also used to determine if new vaccine strains can be neutralized by the antibodies generated with old vaccine formulations or if the viruses used in the vaccine formulations need to be updated to new strains.
In a serology assay based on conventional ELISA analysis, disease-specific antibodies in a patient sample bind to immobilized antigens. Antibody binding is detected with a labeled anti-species antibody. This “direct binding” format, while simple to carryout, is often not used for influenza serology because i) conventional direct binding approaches may not differentiate antibody responses to a recent infection from antibody responses to previous influenza infection; and ii) a response in a direct binding assay may not be indicative of the ability of an antibody to prevent influenza infection. To circumvent these problems, most influenza serology measurements are carried out using techniques that focus on identifying antibodies (termed “neutralizing antibodies”) that bind to hemagglutinin or neuraminidase active sites and prevent influenza from binding and/or infecting host cells. Because these active sites change over time as new strains evolve, neutralizing antibodies tend to be more indicative of recent infections. In vaccine studies, the generation of neutralizing antibodies is also more indicative of vaccine efficacy.
During the infection process, influenza viruses bind to sialic acid groups on host cells through a sialic acid receptor, the viral hemagglutinin protein. Hemagglutination inhibition (HAI) assays measure the ability of antibodies to bind virus hemagglutinin proteins and prevent the binding of virus to red blood cells. The assay end point is visual. In the absence of antibody, the binding of the virus to red blood cells causes the formation of a red gel-like aggregate (hemagglutination) that fills the test well/tube. In the presence of a sufficient neutralizing antibody, hemagglutination is prevented and the red blood cells settle to a small pellet at the bottom of the test well/tube. Microneutralization assays involve mixing virus with the test antibody sample and combining that mixture with a virus-susceptible cell line. In the absence of antibody, the cells become infected while in the presence of sufficient neutralizing antibody the cells remain uninfected.
Both the HAI and microneutralization assays require fresh living cells that must be collected or grown just in time for the assay. These approaches are cumbersome and can lead to significant day-to-day variability in results. Because the end points are binary (hemagglutinated or not; infected or not), a large number of dilutions must be run for each sample to identify the concentration of a sample needed to cause neutralization. The formats are also inherently singleplex. To test the ability of a sample to neutralize multiple different virus strains requires multiple independent measurements.